Part:BBa_K1100121

J23101-ALeader

Background

Riboswitches are regulatory RNAs that regulate gene expression by binding small molecule metabolites. Recently, a novel riboswitch was reported which is present in the leader RNA of the aminoglycoside resistance genes that encode the aminoglycoside acetyl transferase (AAC) and aminoglycoside adenyl transferase (AAD) enzymes. When the aminoglycosides bind to the leader RNA, a conformational change will be induced, leading to the expression of the aminoglycoside resistance genes(Figure 1). (Jia X et al, 2013.) In addition, the leader RNA encompass an integrase site (attl1)which is overlapped by a short peptide encoded by an ORF (ORF11). ( Roth A et al.) Therefore, Aleader is a complicated riboswitch contains ORF11, cistron, aminoglycoside-binding domain and att1 recombination site. Due to the complicacy of Aleader, the secondary structure of it could not be predicted by most software such as Mfold and NUPACK. (Jia X et al, 2013.).

Figure 1. Induction of Aminoglycoside Resistance Genes by Leader RNA-Antibiotic Binding(Jia X et al, 2013.).

The 75nt RNA sequence of ALeader contains two SD sequences (ribosome binding sites) and an anti-SD sequence (CUUC) which can complementarily pair with either of the SD sequences. In the absence of aminoglycosides, anti-SD pairs with SD2. The binding of ribosomes to SD1 triggers the translation of a small peptide which stops at the stop codon ahead of SD2, therefore inhibits the translation of the gene after SD2. When aminoglycosides (kanamycin for example) exists, it will induce a structural change of Aleader. The anti-SD sequence pairs with SD1, consequently unmasking SD2 for ribosomal binding, which results in the translation of the following gene.

Our Aleader riboswitch demonstrates progressive induction of reporter genes in response to sublethal doses of the antibiotics. It makes sense because an antibiotic-resistant riboswitch must be able to detect low levels of antibiotics and activate the resistance mechanism before the cells are killed. Thus, Aleader turns out to be a novel translation regulatory part with high dynamic range, slight response delay and immense modification potential.

Quantitative Measurement

We tested the ALeader performance by measuring the fluorescence intensity of mRFP1 when different concentrations of kanamycin sulfate are added to the bacteria culture.

Figure 2. The dose-response curve of ALeader with the promoter J23101, measured by mRFP fluorescence test.

More data? See BBa_K1100000

Reference

[1] Jia X, Zhang J, Sun W, et al. Riboswitch Control of Aminoglycoside Antibiotic Resistance[J]. Cell, 2013, 152(1): 68-81.

[2] Hanau‐Berçot B, Podglajen I, Casin I, et al. An intrinsic control element for translational initiation in class 1 integrons[J]. Molecular microbiology, 2002, 44(1): 119-130.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]